The present invention relates to a method for the use of a phage associated lysing enzyme for the detecting the presence and determining the quantity of bacteria present in or on a wide variety of substances.
Microbial contamination of surfaces and substances is a significant cause of morbidity and mortality. Rapid and routine procedures for quantitative determination of bacteria present on surfaces is frequently of vital importance, particularly in food processing, drinking water, and in hospitals. Food poisoning is often a result of microbial contamination of meat or food that occurs during processing. Contamination can be spread through contact of food with surfaces. In addition, spread of disease in hospitals and other facilities often occurs as a result of passage of infectious microbes on the surface of clothes or equipment.
A key feature of these applications is the requirement for rapid testing within minutes, a method that will overcome the potential contaminants from a variety of surfaces, a requirement for no cross-over in the results from one test to a second, and a need for both general and specific testing for microbes, that is, the ability to test for contamination by both microbial counts and the ability to test for the presence of specific microbes.
Various methods have been utilized to measure microbial contamination on surfaces. Traditional procedures for assaying bacteria on surfaces are based on swabbing the surface followed by a culture of the swab for 24 to 48 hours in or on media that supports the growth of microbial species. The cultures are observed manually or with automated instrumentation to determine the number of colonies that have formed as an indicator of the number of microbes initially present on the surface. The disadvantages of this methodology are long assay times, requirements for specially trained personnel, and possible inadequate identification of the presence of certain potentially pathogenic microbes whose growth is not supported by the specific media or environment. In particular, it may be difficult to detect fungal contamination by this method. In addition, in many of the potential applications, the method does not give results in the time frame required for effective response.
Luminescent reactions have been utilized in various forms to detect bacteria in fluids and in processed materials. In particular, bioluminescent reactions based on the reaction of adenosine triphosphate (ATP) with luciferin in the presence of the enzyme luciferase to produce light (the xe2x80x9cfireflyxe2x80x9d reaction) have been utilized. Since ATP is present in all living cells including all microbial cells, this method can be used in a rapid assay to obtain a quantitative estimate of the number of living cells in a sample. Early discourses on the nature of the reaction, the history of its discovery, and its general area of applicability are provided by E. N. Harvey (1957), A History of Luminescence: From the Earliest Times Until 1900, Amer. Phil. Soc., Philadelphia Pa and W. D. McElroy and B. L. Strehler (1949) Arch. Biochem. Biophys. 22:420-433. Alternatively, chemiluminescent detection by isoluminol or similar compounds has been used. This method is based on the detection of iron-containing substances in microbes.
Test procedures exemplifying the use of bioluminescent reactions for bacterial determinations and, specialized instrumentation for measurement of the associated light emission, are known and have been disclosed. Plakas (U.S. Pat. Nos. 4,013,418, 4,144,134, and 4,283,490) teaches a bioluminescent assay for the detection of bacteria in a sample including the steps of lysing non-bacterial cells, effecting filtration by positive pressure, washing, lysing bacterial cells and detecting ATP released with a luciferin/luciferase/Mg2+ regent. The art in this patent does not deal with the specific problems associated with collection of material from a surface or with the detection of specific bacteria. No issue of the timing is mentioned and the invention as disclosed would require significant time.
Chappelle in U.S. Pat. No. 4,385,112 discloses a method for detection of bacteria in water based on bioluminescence. This test requires several hours to perform and is specifically addressed to the detection of total bacterial content in water.
Miller (PCT application U.S. Ser. No. 88/00852) discusses a similar assay for use with urine samples, but does not discuss the issues of collection from a surface and the assay timing is not specifically set forward in this application. further, no method for detection of specific bacteria is elucidated.
U.S. Pat. No. 3,933,592 (Clendenning) discusses a method for bioluminescent detection of microbial contamination and in the examples refers to performing the procedure in less than 2 minutes. The procedure does not involve pre-treatment phases and the removal of somatic cell ATP.
U.S. Pat. No. 5,258,285 (Aegates) discloses a method for detection of bacterial concentration in a sample that utilizes a filtration step, a washing step to remove extraneous material including somatic cell ATP, establishing an extraction chamber in which luciferin/luciferase/Mg2+ is added and the reaction measured. This method does not mention time. In addition, it utilizes separate chambers for washing, extracting the bacterial ATP, and measuring the reaction. This may potentially result in decreased sensitivity due to loss of the material in the process of transferring the solution from chamber to chamber. Further, the method does not describe a means of collecting a sample from a surface.
U.S. Pat. No. 5,736,351 (Miller et al) discloses a method and device for determining the presence and concentration of total microbial contamination or the presence and concentration of a specific microbial species on a surface is described. The method consists of a means of a collection device and fluid for removing the microbes from the surface and suspending them in a fluid phase. An aliquot of the fluid phase is introduced into a disposable test ticket which allows filtration of the sample to remove extraneous substances including somatic cells, and concentration of the microbes. The total concentration of microbes is determined by adding a somatic and bacterial releasing reagent to a disposable test device which comprises a membrane containing the luminescent reagents luciferin and luciferase, and introducing the disposable test device into a luminometer that can read the luminescence from the underside.
The present invention is a method and device for detecting the presence and/or concentration of specific bacteria found on surfaces, in or on food, in the air, water, or in biological samples, by the use of bioluminescence. This device and method relies on the use of at least one lytic enzyme produced by a bacteria infected with a bacteriophage specific for the bacteria to lyse the bacteria, allowing for the release of ATP in the bacteria and its detection by chemical-bioluminescence analysis. Unlike the method and device of U.S. Pat. No. 5,5736,351, the lytic enzyme can be used in place of the bacterial releasing agent, and it may not even be necessary to use the somatic releasing agent, thereby simplifying the procedures.
Reports have described the characteristics of an enzyme produced by the group C streptococcal organism after being infected with a particular bacteriophage identified as C1 (Maxted, W. R. xe2x80x9cThe Active Agent in Nascent Page Lysis of Streptococci,xe2x80x9d J. Gen Micro., vol 16, pp 585-595, 1957, Krause, R. M., xe2x80x9cStudies on the Bacteriophages of Hemolytic Streptococci,xe2x80x9d J. Exp. Med, vol. 108, pp 803-821, 1958) and Fischetti, (Fischetti, V. A., et al, xe2x80x9cPurification and Physical Properties of Group C Streptococcal Phage Associated Lysin,xe2x80x9d J. Exp. Med, Vol 133 pp. 1105-1117, 1971). The enzyme was given the name lysin and was found to specifically cleave the cell wall of group A, group C, and group E streptococci. These investigators provided information on the characteristics and activities of this enzyme with regard to lysing the group A streptococci and releasing the cell wall carbohydrate.
U.S. Pat. No. 5,604,109 (Fischetti et al.)(which is incorporated herein in its entirety by reference) teaches the rapid and sensitive detection of group A streptococcal antigens by a diagnostic test kit which utilizes a sampling device consisting of a throat swab made of synthetic or natural fibers such as Dacron or rayon and some type of shaft which holds the fibers and which is long enough to place the fibers in the tonsillar area and capable of being used to swab the area to remove sufficient numbers of colonizing or infecting organisms. The swab can then be placed in the enzyme extraction reagent in several configurations and subsequently used in an immunoassay. The invention can comprise a test kit for detecting Group A streptococci antigens, comprising an extraction reagent containing lysin enzyme for releasing Group A streptococcal components, and a ligand capable of binding with a component of the Group A streptococcus.
U.S. Pat. No. 5,997,862 (Fischetti, et. al.) and U.S. Pat. No. 5,985,271 (Fischettietal.) disclose the use of an oral delivery mode, such as a candy, chewing gum, lozenge, troche, tablet, a powder, an aerosol, a liquid or a liquid spray, containing a lysin enzyme produced by group C streptococcal bacteria infected with a C1 bacteriophage for the prophylactic and therapeutic treatment of Streptococcal A throat infections, commonly known as strep throat.
U. S. patent application Ser. No. 395,636 (Fischetti, et. al) discloses a method for the prophylactic and therapeutic treatment of bacterial infections which comprises the treatment of an individual with an effective amount of a lytic enzyme composition specific for the infecting bacteria, and a carrier for delivering said lytic enzyme. The methods disclosed included the topical, oral, and respiratory methods of delivering the enzyme. Another method disclosed in that application includes the use of suppositories. These methods and compositions can be used for the treatment of upper respiratory infections, skin infections, wounds, and burns, vaginal infections, eye infections, intestinal disorders and dental problems.
U.S. patent application Ser. No. 395,637 (Fischetti et al.) discloses a method and composition for the topical treatment of streptococcal infections by the use of a lysin enzyme blended with a carrier suitable for topical application to dermal tissues. The method for the treatment of dermatological streptococcal infections comprises administering a composition comprising an effective amount of a therapeutic agent, with the therapeutic agent comprising a lysin enzyme produced by group C streptococcal bacteria infected with a C1 bacteriophage. The therapeutic agent can be in a pharmaceutically acceptable carrier.
In one embodiment of the present invention, the method comprises first collecting a sample in a prescribed fashion using a collection apparatus means comprised of an absorbent or adsorbent material. The collection apparatus means is placed into a container containing a fluid and agitated to release the surface contaminants from the collection apparatus means into the fluid. The collection apparatus means can be in the form of a sponge or a swab and the container can be a bag, tube, or small cup. An aliquot of the fluid phase is subsequently transferred to a disposable test device comprised of a translucent hollow cylinder, open on the top and having a porous filter attached on the bottom. The fluid phase is filtered through the disposable test device comprised of a translucent hollow cylinder, open on to and having a porous filter attached on the bottom. The fluid phase is filtered through the disposable test device by applying either positive or negative pressure resulting in retention of microbes or target analytes on the surface of the filter. The filtration process results in the concentration of an analyte and the removal of any interfering substances from the collectate prior to testing, such as inhibitors or any nonspecific materials to maximize test sensitivity and specificity. The filter retentate can be washed by adding appropriate wash solution and reapplying appropriate pressure to force the fluid phase through the filter. Another feature of the present invention is that the retentate captured on the filter of the disposable test device can be assayed by a chemiluminescent or bioluminescent test method. This final step of the test method comprises adding a luminescent substrate to the retentate resulting in a chemiluminescent reaction and measuring the light output from said chemiluminescent reaction by using a photometer that accommodates the disposable device in a manner which allows its precise and reproducible positioning with respect to the surface of the photosensor and which precludes any possible loss of the final reaction mixture during and after the measurement cycle.
During this procedure, somatic releasing agent may be added to the cells as they are collected on the filter. The filter is washed. Then, 10 to 15 seconds prior to the chemiluminsescent reaction, at least one lytic enzyme produced by a bacteria infected with a bacteriophage specific for that bacteria is applied to the filter.
The present invention allows for a surface contaminant to be identified and/or concentration determined in less than 1 hour from time of collection to end result, and generally in less than 15 minutes.
More specifically, the present invention comprises a method for performing chemiluminescent assays such as bioluminescent assays for ATP, including those utilizing, enhancers, or enzymes such as adenylate kinase, chemiluminescent immunoassay, reflectance, conductive or DNA probe assays. One embodiment of the present invention is a method for determining the microbial contamination by a specific bacteria comprising the steps of:
a) collecting a surface sample with a collection means;
b) agitating said collection apparatus means with a fluid phase to dislodge the surface contaminants into a fluid phase, said fluid phase becoming the collectate;
c) placing an aliquot of said collectate into a disposable test device;
d) applying a positive pressure to the top of the disposable test device or negative pressure to the bottom of the disposable test device to eliminate the liquid phase containing free ATP and any chemical inhibitors as well as concentrating the bacteria at the interface;
e) adding at least one lytic enzyme produced by a bacteria infected with a bacteriophage specific for the bacteria; that lyses the specific bacterial cells present in the aliquot;
f) adding ATP free luciferin and luciferase reagent, and
g) determining the amount of ATP present by measuring the light emitted through translucent sides of said disposable test device.
The step of adding a washing/lysing reagent that lyses any somatic cells present in the aliquot may be included prior to applying the positive pressure to the top of the disposable test device or negative pressure to the bottom of the disposable test device.
The choice of collection fluids are well known to those skilled in the art. Generally the fluid is comprised of a detergent, salt, or buffer or any combination thereof that maintains the integrity of the microbial cell walls. A fluid consisting of 0.15M sodium chloride containing 0.5% Tween 20 detergent is one such choice. It is possible to use other formulations including phosphate or HEPES buffered saline and other detergents including zwitterionic detergents and non-ionic detergents.
It will be obvious to a person skilled in the art that mixing of reactant could be achieved in any of the steps through the use of a micropipette. The detection method of this invention specifically allows for both the concentration of analyte and any resulting chemiluminescent reaction caused by the presence of said analyte to occur within the chamber of the disposable test device. An added feature of the disposable test device is that the diameter of the filter is from 0.5 to 5.0 cm, preferably about 1.0 cm, so that the volume of bioluminescent or chemiluminescent substrate solution is minimized to maximize signal output to the photodetector means. The final volume of the substrate should be between 20 xcexcl to 1000 xcexcl, most preferably about 60 xcexcl to 100 xcexcl. The disposable test device can be inserted into a complementary device comprising a larger (liquid tight) at least two component chamber that can house the disposable test device and through which a volume of collectate greater than 500 xcexcl can pass through the filter under positive or negative pressure and retain the microbes or the analytes of interest onto the surface of the filter. For example, the disposable test device can be inserted into the lower chamber of the two component device, said lower chamber having an outflow for the filtrate to which is attached a removable upper chamber of the two component device. The upper chamber comprising a liquid tight seal to said lower compartment and having an intake valve. Said intake valve can be configured for a complementary Luer tip fitting for attachment of a Luer tipped syringe. Said syringe may include at least one series of prefilter(s) to remove any larger debris from entering the filter of the disposable test device. At completion of passing the collectate through the filter of the disposable test device, the two component device can be opened, and the disposable test device physically removed. Said disposable test device now containing the retentate from a large volume of collectate (i.e. 50 ml). The filtration of said large volume of collectate enables increased sensitivity for analyte detection of the collectate fluid. Said disposable test device is then processed as previously described.
The luciferin/luciferase chemiluminescent reactions for ATP are well known. Other chemiluminescent reactions employing bacterial luciferase reactions, or luminols for total microbial determinations can be easily adapted to the methods and devices of the present invention.
The luciferin/luciferase chemiluminescent reactions for ATP are well known. Other chemiluminescent reactions employing bacterial luciferase reactions, or luminols for total microbial determinations can be easily adapted to the methods and devices of the present invention.
The invention further concerns a detection method in which the presence and quantity of specific microbes on a surface can be detected in a time frame less than one hour, said method comprising the steps of
a) Providing a clean disposable test device comprising an open top, translucent sides and a porous filter attached to the bottom side,
b) Adding an aliquot of collectate, said collectate being that described as above,
c) Adding an appropriate wash solution comprised of detergent, or buffered salts or a combination thereof,
d) Applying positive pressure to the top of the disposable test device, or negative pressure to the bottom of the disposable test device to remove fluid from the device and deposit microbes or target analytes directly or indirectly onto the surface of the porous filter,
e) Adding a specific lytic enzyme directed against the specific microbes to be detected;
f) Adding a chemiluminescent substrate and determining the amount of light emitted by the chemiluminescent substrate using a photometer that accommodates the disposable test device in a manner which allows its precise positioning with respect to the surface of the photosensor and which precludes any possible loss of the final reaction mixture during and after the measurement cycle.
In yet another embodiment of the invention, all of the chemicals and solutions (except for the somatic cell releasing agent and the lytic enzyme) are in a disposable membrane ticket. Such a device is easier to use, particularly in the field, than the use of a disposable test device or large volume concentration device. Fewer liquids need to be added to the membrane ticket, allowing for ease of use, less spillage, a more mobile system and more accurate results. Virtually all of the elements of the invention are essentially self contained in the disposable filter trap.
In yet another embodiment of the invention, all of the chemicals and solutions including the somatic cell releasing agent and the lytic enzyme, are included in the disposable membrane ticket.
The membrane ticket preferably comprises a hinged two sided plastic, cardboard, or paper support having a top and bottom section, and an absorbent pad or disk positioned on top of the inner side of the top section. On top of the absorbent disk is a glass filter membrane, which may be held in place by a plastic or paper rigid layer.
In another embodiment of the invention, the method is used to test bacteria in air samples.
In yet another embodiment of the invention, the method is used to test bacterial contamination of body fluids.
In yet another embodiment of the invention, the method is used to test bacterial contamination of potable water.
In another embodiment of the invention, the method is used to test bacterial contamination of food stuffs.
The bottom section of the membrane ticket preferably comprises a transparent window on the outer side of the bottom section, and a luciferin-luciferase immobilized on the membrane.
Various buffers for extracting antigens and washing immune complexes are well known to those skilled in the art.